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GH70 Dextransucases: Insights on the Molecular Determinants of Dextran

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GH70 dextransucases : Insights on the molecular determinants of dextran molar mass control Marion Claverie To cite this version: Marion Claverie. GH70 dextransucases : Insights on the molecular determinants of dextran molar mass control. Biomolecules [q-bio.BM]. INSA de Toulouse, 2017. English. NNT : 2017ISAT0037. tel-02193638 HAL Id: tel-02193638 https://tel.archives-ouvertes.fr/tel-02193638 Submitted on 24 Jul 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 Last name: CLAVERIE First name: Marion Title: GH70 dextransucrases: Insights on the molecular determinants of dextran molar mass control Specialty: Ecological, Veterinary, Agronomic Sciences and Bioengineering, Field: Enzymatic and microbial engineering. Year: 2017 Number of pages: 232 Glucansucrases (GS) from glycoside hydrolase family 70 (GH70) are -transglucosylases produced by lactic acid bacteria. From sucrose, an economical and abundant agro resource, they catalyze the polymerization of glucosyl residues. Depending on the enzyme specificity, α-glucans vary in terms of size, types of glucosidic bonds and degree of branching and have found multiple industrial applications mainly related to their molar mass (MM). However synthesizing polymers of controlled size with average MM ranging from 1 kg/mol to several millions g/mol and low polydispersity using one single enzyme remains challenging. Indeed, the molecular mechanisms underpinning the control of polymer size have been scarcely explored. To tackle this question, two GSs producing dextran (glucan composed of a majority of α-(1,6) linkages) were selected, and their mode of action explored via biochemical and structural analyses coupled to mutagenesis. The first enzyme selected, called DSR-M synthesizes only low molar mass (LMM) dextran (28 kg/mol) exclusively composed of - (1→6) linkages without any trace of HMM dextran (105 to 108 g/mol). In contrast, DSR-OK (second model), produces the highest MM dextran (>109 g/mol) described to date. Several 3D crystallographic structures of a truncated form of DSR-M (DSR-M2), either free or in complex with its substrate or product (isomaltotetraose) in the domain V or in the active site were solved. Such complexes were never obtained before. Noteworthy, one structure encompassed the most complete domain V reported to date. Analyses of these structures coupled to dextran synthesis monitoring, showed that the LMM dextran specificity of DSR-M2 is explained by a distributive elongation mode due to the weak affinity of its two sugar binding pockets in the domain V which interact with the growing dextran chains and allow the synthesis of dextran longer than 16 kg/mol. 15N1H NMR analyses (HSQC), for the first time performed with such a big protein, further revealed the crucial role of aromatic residues in the catalytic domain for the production of dextran from 2 to 16 kg/mol. In comparison, synthesis of HMM dextran by DSR-OK was shown to be mainly due to the sugar binding pockets of its domain V, ensuring much stronger interactions with growing dextran chains. The role of these pockets was evidenced for both enzymes, their functionality proposed to be linked to the presence of one aromatic stacking residue. Their positioning along domain V relatively to the active site is also important to promote efficient binding. All these findings highlight the cooperation between domain V and the catalytic domain for dextran elongation, offer new perspectives to acquire a deeper knowledge on this interplay and open promising strategies for GH70 enzyme engineering aiming at modulating glucan size. Keywords: Glucansucrase, transglucosylase, GH70, dextransucrase, dextran, glucan binding domain, processivity, distributivity Doctoral school: SEVAB (Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieries) Laboratory: Laboratory of Biosystems and Chemical Bioengineering (UMR CNRS 5504, UMR INRA 792), INSA, Toulouse 2 3 NOM: CLAVERIE Prénom: Marion Titre: Dextransucrases de la famille GH70 : investigations sur les déterminants moléculaires du contrôle de la masse molaire des dextranes produits. Spécialité: Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieuries, Filière : Ingénieurie microbienne et enzymatique. Année: 2017 Nombre de pages: 232 Les glucane-saccharases (GS) de la famille GH70 sont des enzymes produites par certaines bactéries lactiques. A partir de saccharose, substrat renouvelable et peu coûteux, elles sont capables de catalyser la synthèse d’α-glucanes, homopolysaccharides dont les propriétés diffèrent suivant la spécificité de l’enzyme (taille, type de liaisons α-osidiques, degrés de branchement). Les glucanes contenant une très grande majorité de liaisons α-(1,6), appelés dextranes, présentent de nombreuses applications industrielles qui dépendent principalement de leur taille. Cependant, la synthèse directe de dextranes de taille contrôlée (de 1 à plusieurs millions de kg/mol) avec une faible polydispersité et en utilisant une seule enzyme n’est encore pas envisageable. En effet, les mécanismes moléculaires mis en jeu pour le contrôle de la taille des polymères produits n’ont encore été que peu explorés. Dans ce contexte, deux GSs ont été sélectionnées. La première, DSR-M synthétise uniquement des dextranes de faible masse molaire (MM) (28 kg/mol) exclusivement composés de liaisons α-(1,6). A contrario, le second modèle, DSR-OK produit le plus long dextrane décrit à ce jour (>109 g/mol). La caractérisation biochimique et structurale ainsi que la construction de mutants ont permis l’exploration du mode d’action de ces deux candidats. Plusieurs structures 3D de DSR-M2 (forme tronquée de DSR-M) - sans ou en complexe avec son substrat ou ses produits (isomaltotetraose) - ont été résolues. C’est la première fois que de tels complexes sont décrits et l’une de ces structures présente le domaine V le plus complet décrit à ce jour. L’analyse de ces structures couplée au suivi cinétique de la synthèse du polymère ont montré que la spécificité de DSR-M pour la synthèse de dextranes courts s’explique par un mode d’élongation distributif dû à la faible affinité de deux poches à sucre de son domaine V envers la chaîne en cours de synthèse. Des analyses RMN (15N1H – HSQC) – jamais réalisées auparavant sur une protéine si grosse – ont également étayé l’importance de la présence de résidus aromatiques dans le domaine catalytique pour la synthèse de dextranes supérieurs à 2 kg/mol. En comparaison, la synthèse de dextranes de haute MM par DSR-OK est principalement due au plus grand nombre de poches à sucre de son domaine V, permettant d’assurer une meilleure interaction avec la chaîne en cours d’élongation. L’implication de ces poches dans la détermination de la taille du dextrane a été montrée pour les deux candidats. Leur fonctionnalité est fortement liée à la présence d’un résidu aromatique de stacking, et leur répartition le long du domaine V a aussi une influence. L’ensemble de ces résultats démontre la coopération du domaine V avec le domaine catalytique pour l’élongation des dextranes, tout en offrant de nouvelles perspectives pour approfondir la compréhension de ce mécanisme. Ils offrent également des stratégies prometteuses pour l’ingénierie d’enzyme de la famille des GH70 pour la modulation de la taille des glucanes. Mots Clés : Glucane-saccharase, dextrane-saccharase, GH70, transglucosylase, dextrane, domaine de liaison au glucane, processivité, distributivité Ecole doctorale: SEVAB (Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingénieries) Laboratoire: Laboratoire d’Ingénieurie des Systèmes Biologiques et des Procédés (UMR CNRS 5504, UMR INRA 792), INSA, Toulouse 4 5 Publications Investigations on the determinants responsible for low molar mass dextran formation by DSR-M dextransucrase. Claverie M., Cioci G., Vuillemin M., Monties N., Roblin P., Lippens G., Remaud- Simeon M. and Moulis C. ACS Catal., 2017, 7, 7106–7119 Futile cycle engineering of the DSR-M dextransucrase modifies the resulting polymer length. Claverie M., Cioci G., Guionnet M., Schörghuber J., Lichtenecker R., Moulis C., Remaud-Simeon M. and Lippens G. In preparation High molar mass dextran synthesis by DSR-OK dextransucrase from Oenococcus kitaharae DSM 17330, role of the domain V? Claverie M., Cioci G., Bondy P., Vuillemin M., Esque J., Magali Remaud- Simeon and Claire Moulis. In preparation Characterization of the First α-(1→3) Branching Sucrases of the GH70 Family. Vuillemin M., Claverie M., Brison Y., Séverac E., Bondy P., Morel S., Monsan P., Remaud-Simeon M. and Moulis C. (2016). J. Biol. Chem. 291, 7687–7702. A dextran with unique rheological properties produced by the dextransucrase from Oenococcus kitaharae DSM 17330. Vuillemin M., Grimaud F., Claverie M., Rolland-Sabaté A., Garnier C., Lucas P., Monsan P., Dols-Lafargue M., Remaud-Siméon M., and Moulis C. (2017). Carbohydr. Polym. 179, 10- 18. Oral communications Discovery of new -transglucosylases from Leuconostoc citreum NRRL B-1299 and NRRL B -742 for the synthesis of tailor-made -glucans. Claverie M., Vuillemin M., Passerini
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  • Crystal Structure of Glucansucrase from the Dental Caries

    Crystal Structure of Glucansucrase from the Dental Caries

    5 Life Science PF Activity Report 2010 #28 Crystal Structure of Glucansucrase from the Dental Caries Glucansucrases are members of the glycoside hy- second sucrose binding site, namely, subsite +1 and Pathogen, Streptococcus Mutans drolase family 70, and catalyze the formation of glucan +2 (Fig. 1(b) and 1(c)). Trp517 provides the platform with various types of glucosidic linkages, (1-3), (1- for glycosyl-acceptor binding, whereas residues such 4) or (1-6) bonds, from sucrose via transglycosylation as Tyr430, Asn481, and Ser589 comprising subsite lucansucrases from Streptococcus mutans (GTF-SI) catalyze an essential factor in the pathogenesis of dental reactions. In the oral cavity, glucan synthesis by S. mu- +1 are conserved in glucansucrases but not in sugar- caries. Resolution of the GTF-SI structure confi rmed that the domain order of glucansucrase-SI was circularly tans involves three extracellular enzymes, GTF-I, GTF- cutting enzymes. Among these residues, the position Gpermuted compared with that of the well-known -amylase, which catalyses the breakdown of starch into sug- SI and GTF-S. GTF-I and GTF-SI synthesize mainly in- of Asp593 in GTF-SI is critical for glucansucrases that ars. Based on the structure of GTF-SI and a comparison of the amino acids of other glucansucrases, it was revealed soluble sticky glucan with (1-3) glycosidic linkages. We make insoluble and sticky glucan with (1-3) glycosidic that the position of Asp593 in glucansucrase-SI is the most critical point for the orientation of the acceptor sugar, and have used AR-NE3A and 5A beamlines to identify the linkages.